Our long-term goals are to develop clinically safe human butyrylcholinesterase (huBuChE) variants as[unreadable] hydrolytic catalysts to protect populations at risk from exposure to chemical warfare agents or[unreadable] organophosphate (OP) pesticides. The challenge to convert huBuChE into an OP hydrolytic catalyst is to[unreadable] identify huBuChE variants that are resistant to inactivation, catalyze OP hydrolysis efficiently and rapidly and[unreadable] spontaneously dephosporylate. The underlying hypothesis for our work is that combinations of huBuChE[unreadable] mutations at multiple residues, close to and/or far from the active site, are needed to markedly improve the[unreadable] OP hydrolysis activity to ultimately serve as a clinically useful hydrolytic catalyst. The primary goal of the[unreadable] current work is to apply powerful molecular evolution strategies to produce huBuChE variants with[unreadable] significantly improved hydrolytic activity for OP nerve agents. The Aims for this research include: 1)[unreadable] Stereoselective synthesis of different classes of enantiomerically pure OP model substrates for functional[unreadable] screening; 2) Optimization and validation of the functional screening system with a site-saturation mutation[unreadable] library; 3) Evolution of huBuChE variants with catalytic activity using two model compounds representing[unreadable] tabun and soman; 4) Evolution of huBuChE variants with broad specificity; and 5) In vitro and in vivo[unreadable] efficacy testing of evolved huBuChE candidates using authentic nerve agents in collaboration with the[unreadable] Center. This work provides the research and development (R&D) effort required to identify OP catalytic[unreadable] huBuChE variants through a combinatorial approaches including molecular evolution, rational designed[unreadable] mutagenesis, in vitro and in vivo efficacy functional screening. Upon completion of this study, we will have[unreadable] the third generation huBuChE products ready to enter advanced development including production under[unreadable] good manufacturing practice (GMP) conditions, advanced pharmacological screening, and preclinical testing,[unreadable] a process that has been successfully established to transition two generations of huBuChE based protein[unreadable] drugs, human plasma derived huBuChE and recombinant wild-type huBuChE, for advanced product[unreadable] development.